Mass spectrometric diagnosis of septicemia

ABSTRACT

The invention mainly relates to the mass spectrometric identification of pathogens in blood cultures from bloodstream infections (septicemia). The invention provides a method with which microbial pathogens can be separated in purified form from blood after a relatively brief cultivation in a blood culture flask, without any interfering human proteins or any residual fractions of blood particles such as erythrocytes and leukocytes, and can be directly identified by mass spectrometric measurement of their protein profiles. The method is based on the use of relatively strong tensides to destroy the blood particles by dissolving the weak cell membranes and most of the internal structures of the blood particles; in spite of the fact that tensides are regarded as strong ionization inhibitors in MALDI and other ionization processes required for mass spectrometric measurements. This method allows unknown pathogens to be obtained in their pure form by centrifuging or filtration and to be identified on the taxonomic level of species or subspecies. Problems with DNA from high levels of leukocytes can be resolved by special measures. After sufficient cultivation, the identification in a mass spectrometric laboratory takes only half an hour.

PRIORITY INFORMATION

This patent application is a continuation of U.S. patent applicationSer. No. 14/064,905 filed on Oct. 28, 2013, which is a continuation ofU.S. patent application Ser. No. 13/322,363 now issued as U.S. Pat. No.8,569,010, which claims priority from PCT patent applicationPCT/EP2010/060098 filed Jul. 14, 2010, which claims priority to Germanpatent application 10 2009 033 368.1 filed Jul. 16, 2009, both of whichare hereby incorporated by reference.

FIELD OF INVENTION

The invention relates to the mass spectrometric identification ofpathogens in blood cultures from bloodstream infections (septicemia).

BACKGROUND OF THE INVENTION

Many species of microorganism (termed microbes below), particularlyincluding bacteria and single-cell fungi (as yeast or mold), but alsoalgae and protozoa, can be identified mass spectrometrically with a highdegree of certainty by breeding a colony in the usual way on a nutrientmedium, then transferring small quantities of microbes from the colonyto a mass spectrometric sample support plate, and measuring a proteinprofile directly with a mass-spectrometer. The mass spectrumparticularly represents the masses and abundances of the differentsoluble proteins which are present in sufficiently high concentration inthe microbes. This protein profile of the microbes is used to determinetheir identity by a similarity analysis with reference spectra from aspectrum library.

Specialized mass spectrometers for this purpose and correspondingevaluation and similarity analysis programs are commercially available.At present, this identification procedure proves to be highly successfuland rapidly conquers the microbiological laboratories all over theworld.

“Identification” of the microbes means categorizing them into thetaxonomic hierarchical classification scheme: Domain (eukaryotes andprokaryotes), kingdom, phylum, class, order, family, genus, species andsubspecies. The identification of a microbe sample involves determiningat least the genus, usually the species, and if possible the subspeciesor even the strain as well, which is important, for example, ifdifferent subspecies or strains have different pathogenicity. In a moregeneral sense, an identification can also mean a characterization interms of other, more individual characteristics of the microbes, forexample the resistance of a microorganism against antibiotics.

The nutrient medium for the cultivation of a colony is usually containedas an agar in a Petri dish, which normally results in the growth of purestrains in the form of separated microbe colonies in around six hours tosome days, depending on the reproductive power of the microbes. If thecolonies superimpose or strongly mix, pure colonies can be obtained in asecond cultivation, again carried out in the usual way. In a most simplemethod, some microbes transferred from a selected colony to the massspectrometric sample support with a small swab are then sprinkled with astrongly acidified solution of a conventional matrix substance (usuallyα-cyano-4-hydroxy cinnamic acid, HCCA, or 2,5-di-hydroxy-benzoic acid,DHB) for an ionization by matrix-assisted laser desorption (MALDI). Theacid (usually formic acid or trifluoroethanoic acid) attacks the cellwalls, and the organic solvent (usually acetonitrile) of the matrixsolution can penetrate into the microbial cells and cause their weakenedcell walls to burst. The sample is then dried by evaporating thesolvent, which causes the dissolved matrix material to crystallize.Soluble proteins and, to a much lesser extent, other substances of thecells are embedded into the matrix crystals.

The matrix substance crystals with embedded analyte molecules are thenbombarded with focused flashes of UV laser light in a mass spectrometer,creating ions of the analyte molecules in the hot plasma of the vaporcloud; these ions can then be separated according to their mass andmeasured in the mass spectrometer. Specialized MALDI time-of-flight massspectrometers (MALDI-TOF-MS) are normally used for this purpose. Themass spectrum is the profile of the mass values of these analyte ions.These are quite predominantly protein ions, the ions with most usefulinformation having masses of between 3,000 daltons and 15,000 daltonsapproximately. The protein ions in this method are predominantly onlysingly charged (charge number z=1), which allows for simply referring tothe mass m of the ions instead of always using the term “mass-to-chargeratio” m/z, as is actually necessary and conventional with other typesof mass spectrometry.

The profile of the proteins is very characteristic of the microbespecies in question because each microbe species produces its own set ofgenetically predetermined proteins each having its own characteristicmolecular mass. The abundances of proteins with higher concentrationswhich can be detected by mass spectrometry, are also widely geneticallycontrolled and depend only slightly on the nutrient conditions ormaturity of the colony. The protein profiles are similarlycharacteristic of the types of microbes as fingerprints arecharacteristic of individual humans. Nowadays, reliable and validatedlibraries with well documented reference mass spectra of microbes arebeing thoroughly extended by many laboratories in public and privateresearch institutions and in microbiological institutes of universities.The reference libraries must fulfill strong requirements to be medicallyand legally admissible.

This method of identification has proven to be extraordinarilysuccessful. The certainty of a correct identification is far greaterthan that of the microbiological identification methods used until now.It has been possible to prove that the certainty of the identificationwas way above 95 percent for hundreds of different types of microbe. Inmost cases of doubt, where there were some deviations from results ofmicrobiological identification methods used until now, geneticsequencing has confirmed the correctness of the mass spectrometricidentification.

If the library does not contain a reference mass spectrum for a speciesof microbe under investigation (which occasionally happens due to themillions of microbe species and the limited size of the current spectrallibraries), library searches can usually produce valuableclassifications into the higher taxonomic levels of the genus or familyof the microbes, because related microbes frequently contain a number ofidentical types of protein. These cases are increasingly rare, however;pathogenic microbes have now practically all been recorded in the formof reference spectra and can therefore usually be accurately identifieddown to the level of microbe species.

The method briefly described above of using a small swab to spread somemicrobes from a colony onto a sample spot of a mass spectrometric samplesupport which is then sprinkled with a matrix solution, is the simplestand, as yet, fastest type of sample preparation. If a colony is justvisible after cultivation, it takes only one to two hours in maximumuntil the identification is complete even if hundreds of samples have tobe analyzed at the same time. Mass spectrometric sample supports with48, 96 or 384 sample spots each are commercially available; acquisitionof mass spectra from these numbers of samples takes around half an hourto two hours. If the identification is urgent, individual microbesamples can be identified in a few minutes (albeit after cultivation,which is always time-consuming).

Other methods of sample preparation have also been investigated, such asextracting the proteins after the microbes have been destroyed bysonication or mechanical treatment, or methods for extracting theproteins from microbes after the cell walls, which are sometimes hard,have been weakened by aggressive acids. These disintegration methods areused when the normal method of swabbing fails because the cell walls ofthe microbes are not destroyed by sprinkling with the matrix solution.If the swab methods produce mass spectra which are good enough for acomparison, all the disintegration methods provide spectra which arevery similar to those of the swab methods, they often even show a lowerinterfering background in the mass spectra.

Today, mass spectra of the microbe proteins are usually acquired in thelinear mode of MALDI time-of-flight mass spectrometers (MALDI-TOF)because these have a particularly high detection sensitivity, eventhough the mass resolution and the mass accuracy of the spectra fromtime-of-flight mass spectrometers in reflector mode are much better. Inreflector mode, only around one twentieth of the ion signals appear,however, and the detection sensitivity is one or two orders of magnitudeworse. The high sensitivity is based on the fact that not only thestable ions but also the much more abundant fragment ions and even theneutral particles from a so-called “metastable” decay which occursduring the flight of the ions, are detected in the linear mode of atime-of-flight mass spectrometer. Secondary electron multipliers (SEM)are used as ion detectors, measuring molecular ions, fragment ions andneutral particles because they all generate secondary electrons onimpact. All fragment ions and neutral particles generated afteracceleration in the ion source from one species of parent ion have thesame speed as the parent ions and thus arrive at the ion detector at thesame time. The arrival time is a measure of the mass of the ions whichwere originally undecomposed.

The increased detection sensitivity is so crucial for many applicationsthat one accepts many of the disadvantages of operating thetime-of-flight mass spectrometers in linear mode, such as asignificantly lower mass resolution, for example. The energy of thedesorbing and ionizing laser is increased for these applications,something which increases the ion yield but also increases theirinstability, although this is of no consequence here.

Acquiring mass spectra with time-of-flight mass spectrometers generallyrequires that a very high number of individual spectra are measured anddigitized in rapid succession, the individual spectra usually beingadded together by adding measurement points with the same time-of-flightto form a sum spectrum. The ions for each individual spectrum aregenerated by one laser flash from a UV pulse laser for each spectrum.The sum spectra have to be generated in this way because of the lowdynamic range of measurement and high noise on the signal in theindividual spectrum. A minimum of approximately 50, in some cases even1,000 and more individual spectra are acquired here; a sum spectrumgenerally consists of several hundred individual spectra which modernmass spectrometers acquire and add together in a few seconds.

For the identification of microbes, usually mass spectra from around2,000 daltons to high mass ranges of 20,000 daltons are measured.However, mass signals in the lower mass range up to around 3,000 daltonsdo not have a high degree of reliability because they originate to alarge part from coating peptides externally attached to the microbes,from fatty acids depending on kind and availability of nourishment, anda variety of other substances which are present only by chance. The bestidentification results are obtained if only the mass signals in the massrange between 3,000 and 15,000 daltons are evaluated. The low massresolution, which occurs for the reasons given, means that the isotopegroups whose mass signals each differ by one dalton can no longer beresolved in this mass range. The mass signals, therefore, reflect theshape of the envelopes of the isotope groups.

This method of identifying microbes requires a pure culture of identicalmicrobes, a so-called “isolate”, in order to obtain a mass spectrumwhich is not superimposed with signals of other types of microbes. Itturned out, however, that mass spectra of mixtures of two microbespecies can also be evaluated by special methods and that both microbespecies can be identified. The identification certainty suffers onlyslightly. If more than two microbe species contribute to the massspectrum, the identification probability and identification accuracydecrease very strongly.

The identification of microbes is particularly important for infectiousmicrobes within the blood stream, called septicemia. The microbesusually are released into the blood continuously or in batches fromunknown focuses of infections. It is important here that pathogenspecies are identified very early to commence a targeted medicaltreatment with correct antibiotics as soon as possible.

The mass spectrometric identification method competes with PCR analysis,where certain genetic sequences of the microbe's DNA, which arecharacterized by selectively operating pairs of primers, are replicatedby polymerase chain reaction (PCR). These methods are fast and can leadto results within a few hours. These PCR analytical methods, however,need some prior knowledge of species, genera, families or classes of themicrobes in order to select correct primer pairs. In general, only a“coarse” classification is performed according to the characteristicsgram-positive or gram-negative, for example. The determination on thelevel of a microbe species is only possible in individual cases andrequires a very targeted approach based on assumptions. The method isusually limited to individual, frequently occurring and particularlydangerous pathogens such as Staphylococcus aureus, for example. Positiveidentifications of individual microbe species remain valuable luckystrikes. In cases of negative identification, the knowledge of theexclusion of such dangerous microbes is certainly also valuable, butdoes not provide a basis for a therapy. The accurate determination ofthe microbe species must then be left to the conventionalmicrobiological methods, which can quite easily take three to five days,however.

German Patent Application DE 10 2007 058 516 A1 (WPO 2009/065580 A1)discloses a method that directly separates pathogens from body fluids bycentrifugation or filtration so that microbes can be transferred fromthe deposits (centrifugation or filtration pellets) onto the samplesupport plate. A still better method, also described in the document, isto disintegrate the microbes of the deposits after removal of thesupernatant still within the centrifugation tube, for example by addinga few microliters of a strongly acidified matrix solution, withsubsequent transfer of the solution with the released proteins onto themass spectrometric sample support plate. With centrifugation, thisdisintegration is even possible when no visible pellet is produced. Thelimit of visibility for a centrifugation pellet is around 10⁶ microbes;the detection limit, in contrast, is currently about 10⁴ microbes, butmay be still improved in the future. 10⁴ microbes usually contain morethan 100 picograms of soluble proteins, the mass spectrometric detectionlimit is far below this, however. Since a cultivation stage is notrequired for infections in clear body fluids, identification in the massspectrometric laboratory with this method of direct centrifuging of thebody fluids can performed within a few minutes.

This method of direct centrifugation or filtration of the microbes issuccessful because, in the vast majority of cases (far more than 70percent), acute microbial infections in body fluids are caused by onlyone single microbe species. At a low percentage of around 15 percent twomicrobe species are involved, in these cases usually both can berecognized in the mass spectra. This species purity of the pathogens ofacute infections is in sharp contrast to other occurrences of microbesin or on the human body—the approximately 10¹⁴ bacteria of theintestinal flora in a human intestine comprise at least 400 species ofbacteria which live in equilibrium with each other, for example. But themethod of direct centrifugation is only successful if these microbes arepresent in very high concentrations of more than 10⁴ microbes permilliliter which is very rare for internal body fluids. Body fluids aregenerally sterile, i.e., they usually do not contain any microbes.

As described in the cited document, this method of sedimenting themicrobes in a centrifuge or micro-filter can be applied directly andwith a high degree of success to all clear body fluids such as lymph,synovial fluid or cerebrospinal fluid (liquor) and even to excreted bodyfluids such as urine or lachrymal fluid. For body fluids containingendogenous particles, such as whole blood, for example, intermediatesteps must be introduced of first growing the microbes by cultivatingthe blood, because the concentration of microbes are usually only in therange of 0.5 to 10 microbes per milliliter, and second to completely andthoroughly destroy the blood particles such as erythrocytes orleukocytes to thoroughly get rid of all human proteins. In the documentscited, this destruction is exemplarily done by addition of distilledwater. The very delicate cell membranes of the human particles areeasily destroyed by the osmotic pressure of the water entering, incontrast to the much harder cell walls of bacteria. By repeated additionof distilled water and centrifugation a sufficiently clean pellet shouldbe obtained which should no longer contain any residues of the humanparticles.

However, experiments carried out in the applicant's laboratory with thismethod of adding distilled water did not succeed in providingsufficiently clean centrifuge deposits, even when the washing andcentrifuging processes were repeated a few times. Even if the depositsdid not retain a slightly reddish color, superimpositions with humanprotein signals always interfered with the microbial protein signalsfrom these deposits to such an extent that an identification becomesuncertain.

There is a need for a fast method for the clean separation of thepathogens of a septicemia in blood, without any traces of remaininghuman proteins, so that mass spectrometric identification down to thelevel of species or subspecies and, in addition, other investigations ofthe pathogens become possible.

SUMMARY OF THE INVENTION

The invention is based on the method for the direct deposition throughsedimentation or pelleting of the microbes from body fluids bycentrifugation or filtration known from German Patent Application DE 102007 058 516 A1 (WPO 2009/065580 A1), but uses a solution of a strongtenside, i.e., an amphiphile, surface-active substance rather than puredistilled water for the destruction of the blood particles. Sodiumdodecyl sulfate (SDS), a strong anionic tenside which is also used inbiotechnology as a denaturizing agent for proteins, has proven to beideal, in spite of the facts that (1) traces of tensides are known to beionization inhibitors for proteins and other analyte substances inionization processes like MALDI, and (2) that strong tensides like SDSare known to kill microbes, i.e., they act bactericidal by destroyingtheir reproduction capability.

In one embodiment, the tenside is added directly and without any furtherpre-treatment to around one milliliter of blood from a blood culture,and mixed well for ten to thirty seconds in a shaker. Preferably thetenside is added as a solution, for example in the form of 10 μl to 200μl of a 5 to 20% aqueous SDS solution. Immediately after mixing, themixture is centrifuged for two minutes at 10,000 g and the supernatantis discarded. If the pellet is visible at all, the deposit shows asurprisingly clear white in most cases. To remove any traces of thetenside, the pellet is washed with a milliliter of distilled water andcentrifuged again. The supernatant is removed again. The deposit can nowsimply be taken up with a few microliters of formic acid andacetonitrile and one to two microliters of the solution can betransferred to the mass spectrometric sample support where matrixsolution is added, and dried.

Since all these preparatory steps can be carried out very quickly,sample preparations on the sample support plate are available for massspectrometric measurements after fifteen minutes at maximum. Theintroduction of the sample support plate into the evacuated ion sourceof the mass spectrometer and the measurement also take only a fewminutes so that the mass spectrometric identification will be availablein less than half an hour. Surprisingly, clean mass spectra of theprotein profiles of the microbes without any interfering signals ofhuman proteins are obtained, and the measurement turns out to be highlysensitive, in spite of the intermediate use of tensides.

This method of sedimenting the microbes from a blood sample for massspectrometric identification can only be carried out if the blood has ahigh microbe level with more than 10⁴ microbes per milliliter; usuallythis is not the case with original blood from a patient. In normalsepticemia, the level of living and active microbes, measured in “colonyforming units” (CFU), is between 0.5 and 10 CFU/ml, only infectedchildren show levels up to and above 100 CFU/ml. The microbes in theblood, therefore, have to be cultivated with suitable additives byincubating the blood in suitable blood culture flasks at optimumtemperature. This cultivation is well-known and significantly fasterthan the cultivation of cultures in Petri dishes and, especially forsevere infections, usually provides sufficient microbes for theidentification within a few hours up to a few days. Only very slowlygrowing microbes, e.g., some mycobacteria, require cultivation times ofmany days to some weeks.

This method has the unique advantage of classifying the microbes withoutany prior knowledge down to the level of microbe species or evensubspecies. No morphological (or microscopic) inspections, biochemicalbasic reactions or other types of pretest are necessary. Knowing themicrobe species, the physician treating the patient can then immediatelycommence a targeted therapy because, for most microbes, information isavailable about the antibiotics to which they respond. The resistancesof this microbe species against antibiotics, which can differ fromregion to region or from hospital to hospital, are usually also known.The early commencement of targeted treatment of a patient who is in anintensive care unit with a septicemia is not only extremely beneficialfor the patient (up to live-saving), but also strongly cost-saving.

Although dissolving blood particles by strong tensides, in particular bySDS, is known in microbiology, the method is usually not applied. U.S.Pat. No. 4,753,749 discloses microbiocidal cleaning agents and teachesthat detergents exhibit a certain amount of microbiocidal activity thatis not sufficiently high to inhibit the growth of all pathogenicmicroorganisms. U.S. Pat. No. 5,336,671 discloses the use of tensidesand defoamers in fungicidal compositions. Strong tensides such as SDSare considered to be bactericidal, and the reproduction capability ofthe microbes is important for microbiological identification methods.This does not apply to the mass spectrometric identification method,however.

The invention is based in particular on dissolving the different typesof blood corpuscles quickly and completely without destroying themicrobes. The delicate cell membrane of the blood particles includespredominantly of phospholipids, which form a membrane by non-covalentbonds. Strong tensides, in particular SDS, dissolve all non-covalentbonds of proteins and lipids and destroy the quaternary and tertiarystructure of the molecules. The cell membranes therefore completelydissolve. The internal structures of the blood particles are alsodestroyed by the SDS and dissolved, including the membrane of the cellnucleus and the chromosomes of the leukocytes. All these dissolvedcomponents are removed with the supernatant fluid after thecentrifugation or by the filtration. Some complications with DNA fromleukocytes are discussed below.

The cell walls of bacteria, on the other hand, are very stable; theyinclude mainly cross-linked polymerized mureins. This covalently boundmesh withstands the dissolving effect of the tensides. For thesubsequent mass spectrometric identification it is not important whetherthe microbes die (for instance by unfolding the tertiary or quaternarystructure of the internal proteins) or remain able to reproduce duringthis method, as long as the proteins in the interior are not lost orchanged in their primary structure. For many microbes, however, thisshort-term separation and isolation method according to the inventionstill leaves sufficient microbes which can be reproduced as isolates ina further cultivation.

These and other objects, features and advantages of the presentinvention will become more apparent in light of the following detaileddescription of preferred embodiments thereof, as illustrated in theaccompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

In FIG. 1, the simple basic procedure is outlined, known to besuccessful for blood of all patients with normal levels of leukocytes.

FIG. 2 shows a method to overcome problems with DNA agglutination forhigh-level leukocytes, but on costs of decreased sensitivity.

FIG. 3 presents a procedure with usual sensitivity which may be appliedto all blood samples with high levels of leukocytes.

DETAILED DESCRIPTION OF THE INVENTION

The preferred embodiments described below are based on blood sampleswhich have a sufficiently high concentration of microbes with more thanabout 10⁴ microbes per milliliter. Only in extremely rare cases, thiscan be the delivered blood in the original state if the physiciancommunicates an extremely acute infection; normally the method relatesto the blood from a cultivation of several hours to a few days inappropriate blood cultivation flasks which are commercially available.The specialist knows, that the cultivation usually is performed forpairs of blood samples in pairs of blood cultivation flasks, one foraerobic microbes and one for anaerobic microbes. The flasks alreadycontain anticoagulants and nutrients; after adding the blood they areincubated it in an incubator at 37° Celsius. In addition, the bloodcultivation flasks already contain inhibitors or adsorption material formost antibiotics. Even if an immediate identification from the originalblood is attempted (which may become possible more often in the futurewhen the mass spectrometric detection sensitivity is sufficientlydeveloped), it is expedient to cultivate the unused portion of theblood. This blood serves as a reserve in case the direct identificationmethod with the original blood does not result in a sufficiently certainidentification.

There are several types of special blood cultivation flasks commerciallyavailable having built-in indicators for a sufficient growth status ofmicroorganisms. In most cases, these indicators are based on ameasurement of the increasing concentration of carbon dioxide producedby the microorganisms. These blood cultivation flasks are easy to handleand indicate automatically sufficient reproduction of microbes by asignal. Investigations in the laboratories of the applicant, however,have shown that this signal comes rather late for mass spectrometricidentifications. Successful identifications can already be obtained withcultivated blood two to four hours before the signal indicatessufficient reproduction of microbes. In cases of dangerous and criticalsepticemia, therefore, it may be opportune to try a mass spectrometricidentification of the microbes in the cultivated blood every one or twohours, or other appropriate time intervals, not waiting for the signal.

An early method of obtaining microbes directly from blood (withoutin-blood culturing) for further cultivation on Petry dishes wasdeveloped by G. L. Dorn around 1976, using lysis of the blood particlesand soft centrifugation. The development of the lysis-centrifugationmethod resulted in the well-known “Isolator™” tubes, made commerciallyavailable first by DuPont (Wilmington, USA), then by WampoleLaboratories, Cranbury, N.J., USA, and nowadays also by Oxoid Limited,Basingstoke Hunt, England. Isolator™ is a trademark of Carter-Wallace,Inc., New York, N.Y. 10105 USA. During the past thirty years, manymillions of Isolator™ tubes were used all over the world to separatemicrobes, particularly including mycobacteria freed from inside humanmacrophages, a kind of leukocytes. According to the “Wampole Isolator™Manual”, the Isolator™ tube contains (a) Saponin for host cell lysis,(b) Polypropylene glycol as foam retardant, (c) Sodium polyanetholsulfate (SPS) as anticoagulant, (d) EDTA as anticoagulant, (e)Fluorinert, a liquid plastic immiscible with water for the concentrationof bacteria (as centrifugation cushion). There are different types ofsaponins, all of them are natural detergents (tensides) generated bydifferent plants. The saponin inside the Isolator™ tube is a detoxifiedsaponin, capable to dissolve the cell walls of the human blood particleswithout killing the microbes. The Isolator™ tubes are used for a softcentrifugation with only 3,000 g for a rather long time of 30 minutes,also with the intention not to damage the microbes. The aim of themethod is the extraction of active microbes from blood for arecultivation of the microbes on suitable nutrients in Petry dishes.

In contrast to this method, the present invention aims for a fastrecovery of purified microbes from cultivated blood for a massspectrometric identification, with the absolute necessity to get rid ofall traces of human proteins which otherwise would disturb theidentification. The reproductive capability of the microbes which isimportant for microbiological identification methods, is not requiredfor the mass spectrometric identification method, because here it isonly the structural integrity of the cell walls and the chemicalintegrity of the internal protein molecules, and not the ability forreproduction which must be retained. For mass spectrometricidentification it does not play any role if internal proteins aredenaturized by unfolding their tertiary and quaternary structure. In thevery fast method according to the invention described above, however,where the microbes are exposed to the SDS for only a few minutes atmaximum, it turns out that many microbes retain their reproductioncapability despite the antibiotic effect of SDS, and so the method ofthe separation and isolation of microbes from blood may also be used toprovide further microbes in isolated form for other investigations like,for instance, resistance tests.

A simple, but already successful embodiment of the method of isolatingand separating the microbes from cultivated blood is presented inFIG. 1. It starts with filling almost one milliliter of blood into eachof several special centrifuge tubes, adding a tenside solution andmixing well. The tenside is preferably added as a solution, in the formof 20 to 200 microliters (preferably 100 microliters) of a 5 to 20percent SDS solution (preferably 10 percent), for example. Fillingseveral centrifuge tubes allows the centrifuge to be balanced and alsomakes it possible to immediately obtain confirmation samples for areliable identification. After mixing for 10 to 30 seconds in a shaker,the blood sample is centrifuged in the centrifuge tubes for two minutesat 10,000 g. The deep-red supernatant liquid is removed, e.g., with aconventional pipette with removable tips. The deposit of microbes is nowtaken up and washed with distilled water, after further centrifugation adeposit of isolated microbes remains which, if visible at all, is purelywhite. Only in critical cases the washing procedure has to be repeatedto get rid of all traces of human proteins; each washing process isprolonging the total procedure by about two to three minutes only.

Handling the SDS solution requires some caution because the solutioneasily foams up, and the stiff foam lasts for hours or even days, makingit hard to use the liquid. In a preferred mode, a foam inhibitor(defoamer) may be added to the SDS solution. There are several types ofdefoamers on the market. The principle of the application of a defoameris already known from Isolator™ tubes.

After the last removal of the supernatant, the soluble proteins of themicrobes have to be extracted. This can be done by a destruction of thecell walls by physico-chemical means, and dissolving the proteins.Usually the extraction is simply performed by addition of around a fewmicroliters of a 70 percent formic acid, which aggressively attacks thepeptidoglycans (mureins) of the cell walls of the microbes and destroysthe cell structure. The same quantity of acetonitrile is then added inorder to dissolve as many proteins as possible. Only in rare cases,other techniques such as for example sonication or mechanicaldestruction are needed. The solution of the soluble proteins iscentrifuged in order to precipitate the solid components such as cellwall fragments, for example, and about one microliter of the supernatanteach is pipetted onto sample spots of a MALDI sample support plate.After drying, around one microliter of a solution of matrix substance,preferably HCCA or DHB dissolved in water and acetonitrile with a smallamount of trifluoroethanoic acid, is added to each of the samplepreparations. All these procedures are be carried out simultaneously forseveral centrifuge tubes which have been filled with blood samples.After drying, the sample preparations on the sample support plate areready for the acquisition of mass spectra. If there are more bloodsamples to be analyzed, these can be worked on simultaneously,depositing the sample preparations on other sample spots of the sameMALDI sample support plate.

There are different types of MALDI sample support plates commerciallyavailable, with 48, 96, and 384 sample spots. There are, for instance,sample support plates which contain hydrophilic anchor places about twomillimeters in diameter in a hydrophobic environment. The transferredsolution then forms a hemispheric droplet two millimeters in diameter onthe anchor place. Other commercial sample support plates containpre-fabricated matrix substance layers (HCCA) on the sample spots.Single-use sample support plates carry visible laser-etched rings withtwo millimeters in diameter which stop the spreading of the droplets.All these sample support plates can be used appropriately for the massspectrometric measurement part of the methods according to theinvention.

The simple preparation method for the measurement samples can bemodified in a wide variety of ways. One option is to use the samplesupport plates which already carry a thin layer of the matrix substance,HCCA, for example. The supernatant of formic acid and acetonitrile isthen pipetted directly onto this thin layer. The thin layer has theproperty of immediately adsorbing all proteins on the surface of thematrix crystals so that after around one minute the remaining liquid canbe removed, e.g. by pipetting or simply with a blotting paper. This alsoremoves impurities like salts or residual tensides. The subsequentoptional addition of a droplet of acetonitrile can embed the proteinsinto the small crystals of the thin layer, thereby increasingsensitivity.

Instead of sedimenting the microbes by centrifugation, they can also bedeposited and washed by micro-filtration. Since the addition of tensidescauses the cell membranes of the blood particles and their internalstructure to practically completely dissolve, a pure isolate of themicrobes is also obtained by micro-filtration.

These processes for separating the microbes from blood and for preparingthe measurement samples take only about 10 to 15 minutes in total. Thesample support plate with the sample preparations is now introduced inthe usual way via a vacuum lock into the ion source of a commerciallyavailable mass spectrometer. The mass spectrometer is operational inaround five minutes. In a mass spectrometer whose UV pulse laseroperates at 200 hertz it takes only a few seconds to acquire asufficient number of individual spectra from a measurement sample inorder to obtain a very usable sum spectrum. The acquisition of the massspectra can therefore be completed after one minute. (Nowadays,MALDI-TOF mass spectrometers are commercially available with 2 kilohertzlasers).

Also commercially available are computer programs for the subsequentidentification of the microbes by their mass spectra, e.g. the“Biotyper” (Bruker Daltonik, Bremen, Germany). The time required for theidentification of the microbes from good mass spectra depends on theperformance of the computer, the size of the library with referencespectra, and the algorithm for the similarity analysis. Withcommercially available computers in mass spectrometers theidentification of the mass spectra from the samples including theconfirmation samples takes only seconds up to a few minutes in maximum;the identification of the microbes is therefore available in digital orprinted form half an hour after the end of a successful cultivation ofthe microbes in blood.

All these simple methods for obtaining pure deposits of microbes andsubsequent identification by mass spectrometry work quite successfulwith blood from normal patients, being delivered the first time into ahospital. The identification success rate of this most simple embodimentof the invention amounts to more than 95 percent.

There are, however, very specialized hospitals of last resort, wherepatients with chronic and widely unknown diseases finally undergothorough investigations and special treatment. In these kinds ofhospital with patients of rare and difficult diseases, up to 40 percentof the blood samples, after being treated with SDS solution, form largemucous cots swimming invisibly in the deep red liquid. These mucousplugs entrain an unknown percentage of the microbes. A skilled personcan pipette some fluid from around the plugs for further treatment, butthis method is difficult and lowers the detection limit by unknownfactors. An investigation suggests that these mucous cots mainly includeDNA from highly increased numbers of leukocytes, possibly intermixedwith coagulated proteins from the blood. The patients usually showhighly increased levels of leukocytes in their blood.

There are several solutions to this problem. As a first and very simplesolution, the blood may be diluted by a factor between two and ten,preferably of about five, with distilled water, before the SDS solutionis added. Diluting by a factor of five increases the success rate tomore than 90 percent, but also decreases the sensitivity of the methodby a factor of five if the same 1 ml centrifugation tubes are used. Incritical cases, the corresponding prolongation of the cultivation timewhich is necessary to produce more microbes, may be unbearable.

As a second solution for the clotting problem, a method of centrifugingthe blood sample before adding the SDS solution can be used. The successrate is improved, probably by removal of all coagulating proteins of theblood before addition of the SDS solution. In this embodiment, which maybe introduced as a standard procedure at special hospitals, the bloodinitially filled into the centrifuge tubes will be centrifuged firstwithout the addition of tenside solution. The supernatant clear bloodplasma with its hundreds of proteins, added nutrients and anticoagulantsis then removed and only the deep-red deposit is taken up with a tensidesolution, a 1 percent SDS solution, for example, filling the tube up toone milliliter in total. The deep-red deposit, which contains not onlythe microbes but normally also the 5 million erythrocytes, 7 thousandleukocytes and 200 thousand thrombocytes from the one milliliter ofblood, is mixed with the added tenside solution in a shaker, therebydestroying the cell membranes of the blood corpuscles and releasing thesoluble proteins. The deep-red solution is now centrifuged again, thesupernatant remaining deep red this time and the deposit, if visible,appearing purely white. The process of removing the supernatant, fillingup with tenside solution and centrifuging may now be repeated to removeeven the last residues of blood particles. The process may then berepeated with pure distilled water to remove the tenside, because itwould interfere with the ionization by matrix-assisted laser desorption(MALDI). After a last removal of the supernatant the deposit, whethervisible or not, is disintegrated as described above and the solubleproteins are transferred onto the sample support plate.

As a third solution for the problem of blood clotting, the SDS solutionmay be prepared with special anticoagulants to inhibit the formation ofthe mucous plugs. This problem solution may be combined with the secondproblem solution.

As a fourth solution, the mucous plugs may be dissolved by addition ofone or more nucleases.

If the deposit is visible after the final washing step, a differentembodiment of the invention can be applied, including the transfer of asmall quantity of the microbes thus isolated with a swab onto the samplesupport plate where they can be prepared as usual. The mass spectra ofthis conventional swab technique are to a large extent similar to themass spectra of the disintegration technique using acid in thecentrifuge tube. If differences are evident here, mass spectra of bothtypes of sample preparation can be entered as reference spectra into thelibrary.

The invention provides a method for the reliable identification ofmicrobial pathogens in blood which is significantly simpler and fasterthan previous microbiological methods, which are practically alwayscarried out via a cultivation of the microbes on gels in Petri dishes.The invention obtains the purified microbes directly from the bloodafter a cultivation procedure, where they are present in sufficientspecies purity, which is not the case for microbes occurring in or onother locations of the human body. In contrast to PCR analytical methodsthe identification can be performed without any prior knowledge andleads directly to an identification on the level of the microbe speciesor subspecies. No other identification method is as fast and reliable.

The invention is based, in particular, on the destruction of thedifferent types of blood corpuscles quickly and completely in such a waythat the microbes are obtained in a very pure form and endogenous bloodproteins from the blood particles do not interfere with theidentification. The structure of the microbes' cells is not destroyed inthis process, in contrast to the blood particles. The delicate cellmembrane of the blood particles includes predominantly of phospholipids,which form a membrane by non-covalent bonds. The effect of the tensides,particularly of SDS, on proteins and lipids is based particularly onbreaking all non-covalent bonds and thus destroying the quaternary andtertiary structure of the cell membrane and cell structure molecules.The cell membranes and all internal cell structures thus dissolvecompletely, the tensides acting as solubilizers. The phospholipids ofthe cell membranes are themselves amphiphilic, i.e., have tensidecharacteristics, and can be nano-colloidally dissolved by other tensidesby forming micelles. The internal structures of the blood particles,including the membrane of the cell nucleus and the chromosomes of theleukocytes, are also destroyed by SDS and widely dissolved. All thesedissolved components are removed with the supernatant after thecentrifugation or by micro-filtration.

The cell walls of bacteria, on the other hand, are very stable; theyinclude mainly covalently cross-linked and thus polymerized mureins(peptidoglycans). For gram-positive bacteria there is an additionalcross-linking with teichoic acids, which are also polymerized. Thesecovalently bound meshes withstand the dissolving effect of the tensidesat least for the short time of several minutes. For the subsequent massspectrometric identification it is irrelevant whether the microbes dieor not in this method, as long as the proteins in the inside are notreleased or changed in their primary structure. For many microbes,however, this short-term method still leaves sufficient microbes aliveand able to reproduce for a further cultivation.

The method is surprisingly simple. The identification of the isolatedmicrobes thus obtained follows conventional methods, which, however,normally are based on an isolation of one type of microbe by growing aseparated colony on a cultivation medium. The isolation here existsautomatically because, for acute infections, only one or at most twospecies of microbe are found as pathogens. This means that separatingthese microbes from infected blood provides quantities of microbes whichrepresent sufficiently pure microbe cultures (isolates). Even when twospecies of microbe are present the method still works satisfactorily.

To simplify the method of identification of microbes in blood, analysiskits with ready-to-use mixtures of tensides and defoamers may beproduced and made commercially available. The mixtures may containadditionally anticoagulants and nucleases; sterile portions of thesemixtures may be contained in ready-to-use evacuated centrifugation cups,easily to be filled with blood samples. The analysis kit may furthercomprise purified solutions for protein extraction from depositedmicrobes, and matrix solutions for the sample preparation on massspectrometric sample support plates. Even one-way mass spectrometricsample support plates may be comprised in this analysis kit.

If the method of separating and isolating microbes is carried out withmore centrifuge tubes than are necessary for the identification, with adozen centrifuge tubes, for example, some of the isolated microbedeposits can also be used for further diagnostic purposes—to determinethe resistance of the microbes, for example, using the conventionalfunctional methods of trial cultivation in the presence of antibiotics.

The method of separating microbe accumulations from endogenous cells cannot only be applied to blood, but also to abscesses or other foci ofinflammation, since they also contain endogenous cells. One example ofthis is a suppurative focus, i.e., an accumulation of some living, somepartially digested microbes in a mixture with certain types ofleukocytes which fight them. In this case, also, the endogenous cellscan be dissolved by tenside solutions. A further example is inflamednasal mucus, which is obtained as a swab of the nasal mucosa and wherethe identification of the microbes is of very great interest. Thesetypes of sample can also be obtained from other mucous membranes.

In the method described above the mass spectra of the microbes wereacquired in mass spectrometers with ionization by matrix-assisted laserdesorption (MALDI). This is the usual way, but not necessarily the onlyone. The solutions of soluble proteins from microbes can also be ionizedby electrospraying, for example. This type of ionization generatesstrong superimpositions of multiply charged ions in the mass range ofabout 600 to 1,600 daltons, which necessarily requires a massspectrometer with high resolution. Time-of-flight mass spectrometerswith orthogonal injection of the ions (OTOF-MS) can be used as the massspectrometer, as can ion cyclotron resonance mass spectrometers (ICR-MS)or other high-resolution mass spectrometers.

The different charge levels of the ions formed by electrosprayionization can be combined mathematically in order to obtain a microbespectrum. It is also possible to conduct a physical charge reduction,however. This involves bringing together positively charged protein ionsand suitable negatively charged ions in an ion reactor located betweenelectrospray ion source and analyzer, resulting in a deprotonation ofthe protein ions. These are introduced into the mass spectrometer, whichmust be able to cope with a large range of masses, however.

Further methods of ionization are also known and can be used here. Anadvantageous method is atmospheric pressure chemical ionization (APCI),for example. The molecules are introduced to the chemical ionization byatomizing a liquid and vaporizing the droplets, or by weak, non-ionizinglaser desorption (“laser ablation”). The chemical ionization suppliespractically only singly charged ions and is thus very favorable, butalso requires a mass spectrometer with sufficiently large mass range.

By knowledge of the invention, the methods described here can bemodified by those skilled in the art in a wide variety of ways. Some ofthese modifications have already been described above; there are alsoadditional methods which can generate the desired informative massspectra of the microbes for their identification on the fundamentalbasis of direct separation of the microbes from blood, abscesses orother inflamed tissue.

Although the present invention has been illustrated and described withrespect to several preferred embodiments thereof, various changes,omissions and additions to the form and detail thereof, may be madetherein, without departing from the spirit and scope of the invention.

What is claimed is:
 1. A method for the mass spectrometricidentification of microbes contained in blood, comprising the steps:providing a blood sample that is suspected of containing microbes,mixing the blood sample with an amphiphile, surface-active substance inorder to destroy blood particles of the blood sample while notdestroying microbial cell structures, depositing the microbes in theblood sample-substance mixture by one of (i) centrifugation and (ii)filtration, acquiring a mass spectrum from the deposited microbes, inthe absence of any prior microbe reproduction step on a flat nutrientmedium, and identifying the microbes down to the species or subspecieslevel by the mass spectrum.
 2. The method according to claim 1, furthercomprising cultivating the blood sample before the mixing in order toproduce a number of microbes in a blood culture sample sufficient formass spectrometric detection.
 3. The method according to claim 1,further comprising washing the deposited microbes to remove remnants ofthe amphiphile, surface-active substance and traces of human proteinsbefore the mass spectrum is acquired.
 4. The method according to claim3, wherein distilled water is used as washing liquid.
 5. The methodaccording to claim 1, wherein the amphiphile, surface-active substanceis anionic.
 6. The method according to claim 5, wherein sodium dodecylsulfate (SDS) is used as the anionic amphiphile, surface-activesubstance.
 7. The method according to claim 1, wherein the amphiphile,surface-active substance is provided as a solution.
 8. The methodaccording to claim 7, wherein the solution contains a defoamer.
 9. Themethod according to claim 1, further comprising centrifuging the bloodsample and removing the resultant supernatant before the mixing in orderto remove liquid blood sample fractions that do not contain anyanalytical information of interest.
 10. The method according to claim 9,wherein the steps of centrifuging the blood sample, removing thesupernatant and mixing with the amphiphile, surface-active substance isrepeated in order to remove the last residues of blood particles. 11.The method according to claim 1, further comprising disintegrating thedeposited microbes by physico-chemical means before the mass spectrum isacquired.
 12. The method according to claim 11, wherein the depositedmicrobes are disintegrated one of (i) physically by sonication ormechanical treatment and (ii) chemically by solutions containing acidslike formic acid or trifluoro ethanoic acid, and organic solvents likeacetonitrile.
 13. The method according to claim 1, further comprisingpreparing measurement samples on a mass spectrometric sample supportplate using a matrix substance in whose crystals the soluble proteins ofthe deposited microbes are embedded before the mass spectrum isacquired.
 14. The method according to claim 1, further comprisingpreparing measurement samples by transferring some microbes of thedeposit onto a mass spectrometric sample support where a matrix solutionis added before the mass spectrum is acquired.
 15. The method accordingto claim 1, wherein the mass spectrum is acquired with a time-of-flightmass spectrometer using one of (i) ionization by matrix assisted laserdesorption (MALDI), (ii) electrospray ionization (ESI), and (iii)atmospheric pressure chemical ionization (APCI).
 16. The methodaccording to claim 1, wherein the microbes are identified using proteinprofiles in the mass spectrum that represent the masses and abundancesof different soluble microbial proteins by means of a similarityanalysis with reference spectra from a spectrum library.
 17. The methodaccording to claim 1, wherein the microbes comprise at least one of (i)bacteria, (ii) single-cell fungi, (iii) yeast, (iv) mold, (v) algae, and(vi) protozoa.
 18. The method according to claim 1, further comprisingagitating the blood sample-substance mixture in order to promotethorough mixing before the microbes are deposited.
 19. The methodaccording to claim 1, wherein the microbes are deposited bymicro-filtration.
 20. The method according to claim 1, wherein theamphiphile, surface-active substance is a denaturizing agent.